- 期刊
與病毒DNA複製無關之假性狂犬病毒轉錄體的選殖與分析
Cloning and Analysis of Pseudorabies Virus DNA Replication-Independent Transcripts
摘要
阿爾伐疱疹病毒的複製生活史可分為即發性早期（α）、早期（β）、早晚期（γ1）及晚期（γ2）等4期。此4期中屬於依賴病毒DNA複製方能表現的僅有γ2時期，且被研究的較為清楚。為了探究與病毒DNA複製無關之假性狂犬病毒轉錄體是由那些時期、那些基因表見所製造的，我們在培養基內添加了泡疹病毒去氧核醣核酸聚合酶抑制劑-phosphonoacetic acid。在假性狂犬病毒感染Madin-Darby牛腎細胞後第6小時（也即是介於β及γ1之轉換點上），萃取細胞總RNA，並以oligo（dT）管柱純化取得poly（A）^+ RNA。以這些poly（A）^+ RNA為模版合成cDNA，並以pUC18選殖載體選殖出1300個含cDNA的選殖株。再以假性狂犬病毒DNA為探針，進行菌落雜交反應，先挑出了10株被假性狂犬病毒核酸探針偵測為陽性的含假性狂犬病毒基因的菌落。再以南方雜交反應定位這10段選殖基因在假性狂犬病毒基因體圖譜上的位置。由計算感染後第6小時cDNA的分布結果顯示，這些轉錄體分布在假性狂犬病毒BamHI第1、2、3、4、5、11及15′片段。同時藉核酸序列的比較下，分析各個cDNA選殖株為何種基因及其出現個數，結果共計篩得3株γ1時期的cDNA（假性狂犬病毒11K基因，假性狂犬病毒主要蛋白衣基因，馬疱疹病毒1型醣蛋白gp13基因相似株），2株β時期的cDNA（假性狂犬病毒43基因，假性狂犬病毒ORF1、2、3基因）及5株未知基因的cDNA。結果證實確實可由病毒感染時間之選擇，配合以疱疹病毒去氧核醣核酸抑制劑之添加，可用以選殖假性狂犬病毒複製時特定時期之表現基因，此選殖途徑可進一步提供特定基因轉錄調節之研究。
並列摘要
Gene expression of α-herpesvirus occurs in a cascade with at least four main phases, commonly referred as immediately early (α)、early(β)、early-late(γ1) and late (γ2). The late genes are expressed after the herpesvirus DNA synthesis and are better understood. In order to study the pseudorabies virus (PRV) DNA replication-independent transcripts, we extract the total RNAs from the PRV-infected Madin-Darby bovine kidney cells at 6 hours post infection (switch point between β and γ1) in the presence of phosphonoacetic acid, an inhibitor of herpesvirus specific DNA polymerase. The poly (A)^+ RNA was purified by oligo (dT)-cellulose spin column and used as a template to synthesize cDNA. The synthesized cDNA was then cloned using pUC18 as a vector. The radiolabeled PRV DNA was used as a probe to screen 1300 cDNA clones by colony hybridization and ten clones containing PRV genetic sequences were obtained. The location of ten cloned PRV cDNA sequences were demonstrated by Southern blot hybridization. These sequences were further analyzed by nucleotide sequencing analysis. The results of Southern blot hybridization indicated that these transcripts are located in the BamHI-1, BamHI-2, BamHI-3, BamHI-4, BamHI-5, BamHI-11 and BamHI-15 fragments of PRV genomes. Comparison of nucleotide sequences indicated that the selected clones contained 3γ1 stage cDNA sequences (PRV 11K, PRV major capsid protein gene, EHV gp13 homologue), 2β stage cDNA sequences (PRV43 gene, PRV ORF1, 2, 3 gene) and 5 unknown cDNA sequences. This experiment proved that the transcripts of PRV could be selected from a specific stage of PRV regulation cycle, and provided a method for study of PRV transcripts.